Since Adagen was approved by the Food and Drug Administration (FDA) as the first protein-polymer conjugate in 1990, the field of protein-polymer conjugates has grown tremendously. Currently, these biological therapeutics have grown to a market of over $7.4 billion in 2011 (Evaluate Ltd. Drug sales database; www.evaluategroup.com). Protein conjugates have diverse therapeutic applications ranging from enzyme replacement therapy to novel functions such as neutralization of over-active cytokines or receptors (Alconcel et al., 2011). As a result, the treatment duration of a protein conjugate also ranges significantly. Some treatments are temporary, but protein-polymer conjugates are also used in enzyme replacement therapy, requiring injections over an extended period to treat chronic diseases such as severe combined immunodeficiency (SCID) or gout (Alconcel et al., 2011).
While protein-polymer conjugates offer unique solutions to problems of missing or malfunctioning enzymes, their chronic use presents long-term challenges in vivo. Currently, all ten Food and Drug Administration (FDA)-approved therapeutic protein conjugates use poly(ethylene glycol) (PEG) (Pfister and Morbidelli, 2014; Besheer et al., 2013; Pelegri-O'Day et al., 2014). PEG is widely used in many disciplines, yet some deficiencies in its therapeutic application have been observed. These include non-biodegradability causing accumulation in tissue and immunological responses such as accelerated blood clearance upon multiple doses (Besheer et al., 2013; Chi et al., 2003).
Polymer conjugation also typically results in a decrease in bioactivity of the conjugate due to steric shielding of the protein active site. In addition, protein therapeutics often must be formulated with excipients for additional stabilization since proteins are highly susceptible to losses in activity when exposed to temperature fluctuations and other stressors (“FDA Access Data”, www.accessdata.fda.gov). While PEGylation often increases stability against environmental stressors, all of protein-PEG conjugates still need to be refrigerated and contain excipients as stabilizers (Leader et al., 2008; Keefe and Jiang, 2012; Nguyen et al., 2013).
PEG alternatives have been developed which improve upon these drawbacks. For instance, previous work in the Maynard group has shown that polymers containing pendant trehalose units stabilize proteins against heat, lyophilization, and electron irradiation (Mancini et al., 2012; Lee et al., 2013; Bat et al., 2015; Lee et al., 2015). Trehalose is a widely used excipient used in the food and cosmetic industries and has been shown to be important in protecting animals and plants against dehydration stress (Jain and Roy, 2009). Other polymers have been shown to exhibit protein-stabilizing properties, including charged polymers, polyols, and other saccharide-based materials (Keefe and Jiang, 2012; Nguyen et al., 2013; Congdon et al., 2013; Stidham et al., 2014; Hu et al., 2015). All these polymers are being actively investigated as PEG alternatives, which also offer stabilization against environmental stressors. However, these examples are still not biodegradable.
Degradable polymers are important to avoid build-up of polymer within the body, especially for enzyme replacement and other chronic therapies. Degradable polysaccharide conjugates have also been prepared by conjugating proteins to biopolymers such as hydroxyethyl starch (HES)(Hey et al., 2012), polysialic acid (Zhang et al., 2010), and dextrin (Hardwicke et al., 2010; Hardwicke et al., 2011). The synthesis of a degradable protein-polymer conjugate by controlled radical polymerization (CRP) has also recently been reported (Decker and Maynard, 2015). Many of these conjugates display increased in vivo half-lives. However, many of these polymers are heterogeneous, which might make FDA approval more difficult, and do not necessarily stabilize proteins.
We sought to prepare well defined polyester backbone and trehalose side chain polymers so that the polymers would stabilize proteins and biodegrade. Previous examples of well-defined biodegradable glycopolymers (none have been reported with trehalose) containing either esters or amides in the main chain backbone were polymerized in two ways: by polymerization of sugar-functionalized monomers, or by post-polymerization modification of polymers containing reactive handles (Xu et al., 2009; Slavin et al., 2011). However, typical polyester or polyamide syntheses require anhydrous conditions, which is compatible with the low solubility of trehalose in typical organic solvents. Therefore, polyesters containing reactive handles were first synthesized, which could be later functionalized with trehalose units after polymerization and purification. While a variety of high-yielding “click” reactions have been demonstrated for the synthesis of glycopolymers, the thiol-ene reaction yields a stable thioether, which can be formed in high yields (Campos et al., 2008).
Polymers may be used as additives to prevent mis-folding and denaturation of proteins. However, the use and development of polymers as food additives and drug component presents its own problems, as polymer longevity causes down-chain problems in waste management and disposal. Due to the wide applicability of polymers in both medical and non-medical fields, interest in developing biodegradable polymers has greatly increased (Agarwal, S. Polym. Chem. 2010, 1, 953-964). Moving towards synthesis of easily degradable, “green” polymers will be increasingly important as polymers continue to be used worldwide.
Trehalose is a non-reducing disaccharide formed by α,α-1,1-linked glucose units, which has been proven to exhibit protection against temperature changes and dehydration2 and is widely used in the food and cosmetic industries. Applicants' previous workhas shown that glycopolymers with pendant trehalose groups offer superior protection to both heat burden and lyophilization, better than free (non-polymeric) trehalose and poly(ethylene glycol) (PEG) (Mancini et al., 2012; Lee et al., 2013). These polymers are promising for a variety of applications, but Applicants herein develop techniques to make the polymers degradable.
Needed in the art are biodegradable polymers that stabilize proteins and biodegrade and that can be readily synthesized with reasonable production. Needed in the art are degradable trehalose glycopolymers that stabilize proteins and other biomolecules (e.g., to the lyophilization process and to heat burden) and also can be degraded through simple processes such as ester hydrolysis.